Aberrant protease activity is essential in many complex tumor processes including growth, invasion and metastasis. Proteolytic events are responsible for unveiling cryptic ECM signaling domains, degrading basement membranes, and activating a suite of molecules including receptors and growth factors. Despite their importance, few technologies exist for detecting and monitoring activities in vivo. Traditional ex vivo techniques like tumor biopsy followed by zymography assays are invasive and provide limited insight since protease activity is highly contextual and tightly regulated in vivo. Recent work with activity-based fluorescent probes are limited by the small number of tissue-penetrating reporters available, precluding studies of proteolytic networks and detection of tumor activity at sites deep within tissue. Here, we propose to circumvent these challenges by constructing long circulating peptide-nanoparticle probes that can survey, sense and remotely report on tumor activity through the urine. In this strategy, iron oxide nanoparticles are utilized as chaperones to deliver protease-specific peptide libraries to tumors whereupon selective cleavage by active proteases releases peptide fragments that are cleared by the renal system into urine. These peptide fragments are predesigned with photo-labile triggers to uncage isobaric peptide "reporters" optimized for multiplexed LC MS/MS quantification. We hypothesize that mass spectrometric analysis of urine in comparative studies involving tumor-bearing and tumor-free mice will uncover unique reporter signatures that can be correlated with protease activity in vivo. Quantified protease signatures will be utilized as a metric for detecting, monitoring and evaluating tumor responses to anti-tumor therapies. This strategy of quantifying proteolytic activity through mass-encoded remote reporters will enable a high degree of multiplexing and will permit the detection of tumors at sites independent of anatomical position. We expect this platform to have broad utility and amenable to a number of protease-dependent diseases such as cardiovascular diseases, coagulopathies, and liver fibrosis. PUBLIC HEALTH RELEVANCE: Non invasive technologies that can detect and profile aberrant enzymatic activity in vivo will have great utility in detecting cancer, monitoring responses to treatment and drug screening. Here we will develop diagnostic nanoparticles to sense and detect protease activity in vivo by monitoring molecular reporters from urine. This approach is broadly applicable to a number of diseases.